Hypothesis

Regulation of Phospholipase C-b1 GTPase-

Activating Protein (GAP) Function and

Relationship to Gq Efficacy

Irene Litosch*

Department of Molecular and Cellular Pharmacology, University of MiamiMiller School of Medicine, Miami, FL, USA

Abstract

How cells regulate Gq efficacy (initiation and termination of Gq

signaling) to effect response remains a central question in

pharmacology and drug discovery. Phospholipase C-b1 (PLC-

b1) is an effector and a GTPase activating protein (GAP) spe-

cific to Gaq. The physiological function of PLC-b1 GAP remains

unclear and controversial. GAPs are generally thought to func-

tion in deactivation of Gq signaling. However, PLC-b1 GAP has

also been shown to increase signaling efficiency through

kinetic coupling with the ligand-activated GPCR. GPCRs func-

tion as guanine nucleotide exchange factors (GEF) on the G

protein activation cycle. This article sets forth a new hypothe-

sis that could unify these conflicting paradigms as it pertains

to physiological signaling and native levels of protein. It is pro-

posed that the physiological function of PLC-b1 GAP is

context-dependent and regulated by phosphatidic acid (PA).

PA stimulates PLC-b1 GAP activity. In the absence of ligand,

PLC-b1 GAP does indeed deactivate Gq signaling, limiting leaky

activation to set the threshold for stimulation to sharpen signal

kinetics. However in the presence of activating ligand, the

increase in levels of PA would stimulate PLC-b1 GAP to kineti-

cally couple with GPCR GEF to increase signaling efficiency.

We found that PA-increased Gq efficiency is dependent on sig-

naling via the unique PLC-b1 PA binding domain. VC 2013

IUBMB Life, 65(11):936–940, 2013

Keywords: G protein; G protein coupled receptor; GTPase activating

protein; phosphatidic acid; phospholipase- b1; phospholipase D

IntroductionThe physiological role of PLC-b1 as a GTPase activating protein(GAP) remains unclear and controversial (1,2). GAPs are gen-erally perceived to limit signal kinetics and signal amplitudefor the Gq/11 (Gq) and Gi/o subfamily of G proteins. GAPs accel-erate the Ga GTPase cycle to shorten the life-span of Gaq-GTP.

Overexpression of GAPs has been shown to deactivate Gq

signaling.Unlike most GAPs, PLC-b1 GAP is specific to Gaq (3). PLC-

b1 is also an effector for Gq. An alternative function for PLC-b1

GAP to increase signaling efficiency has been proposed (1,2).PLC-b1 GAP was found to kinetically couple with ligand-activated GPCR to stabilize the three protein complex consist-ing of m1 muscarinic cholinergic receptor, Gq and PLC-b1, tosustain signal amplitude (4,5).

Which paradigm correctly describes the physiological func-tion of PLC-b1 GAP? The answer to this challenging question isimportant to basic research and drug discovery. Gq is a keyregulator of most human behavior but its potential as a high-value therapeutic target remains to be fully realized (6). Gq

receives and processes information from the ligand-activatedGPCR to implement changes in signaling networks as is neces-sary to achieve appropriate response. PLC-b1 GAP is positionedto determine Gq efficacy and GPCR pharmacology.

This article proposes a new hypothesis that could unifyboth paradigms for PLC-b1 GAP function. It is hypothesizedthat PA stimulates PLC-b1 GAP activity. The physiological

Abbreviations: GAP, GTPase activating protein; GEF, guanine nucleotideexchange factor

VC 2013 International Union of Biochemistry and Molecular BiologyVolume 65, Number 11, November 2013, Pages 936–940

*Address correspondence to: Irene Litosch, Department of Molecular andCellular Pharmacology, University of Miami Miller School of Medicine,Miami, Florida 33101-6189, USA. Tel.: 3052435862. Fax: 113052434555.E-mail: ilitosch@med.miami.eduReceived 4 September 2013; Revised 30 September 2013; Accepted 30September 2013DOI 10.1002/iub.1218Published online 30 October 2013 in Wiley Online Library(wileyonlinelibrary.com)

936 IUBMB Life

function of PLC-b1 GAP is regulated by PA and is dependent onsignaling via the unique PLC-b1 PA binding domain.

The Life Cycle of Gaq-GTP andPLC-b1 GAPGPCRs function as guanine nucleotide exchange factors (GEF) topromote the exchange of cytosolic GTP for the tightly boundGDP on Gaq-GDP of the heterotrimer (7). This rate limiting step inthe G protein cycle generates Gaq-GTP. Signaling is terminated bythe intrinsic Gaq GTPase activity, which converts Gaq-GTP back toGaq-GDP. The intrinsic Gaq GTPase activity is accelerated byGAPs. Overexpression of GAPs inhibits Gq signaling (1,2).

PLC-b1 GAP however can also increase signaling efficiency(4,5). PLC-b1 GAP was found to co-ordinate with the ligand-activated m1 muscarinic cholinergic receptor to regulate theamplitude of the PLC-b1 signal. Essentially, PLC-b1 GAP andGPCR GEF worked as a team to maintain a fraction of Gaq inthe active GTP-bound form to stabilize the complex of GPCR,Gq, and PLC-b1. PLC-b1 GAP was subsequently shown to poten-tiate GPCR GEF activity to increase the rate of dissociation ofbound-GDP, the rate limiting step in G protein activation (8).

Residues required for high affinity Gaq stimulation, GAPactivity, maximum activity and association with membranehave been localized to the PLC-b C-terminal extension, whichcontains a coiled-coil domain (9–14). This domain is unique tothe PLC-b1–4 family. PLC-b also have a conserved core, com-mon to all inositide-specific PLCs, that contains the pleckstrinhomology domain, four tandem EF hand domains, the triosephosphate isomerase barrel-like catalytic domain and a C2domain (15,16).

The crystal structure of a truncated PLC-b3 in complex withactivated Gaq revealed that the Gaq Ras-like GTPase domainmakes multipoint contacts with the enzyme (17). PLC-b3 GAPwas found to depend on residues within a loop off the EF hand.The Gaq construct was truncated at the amino terminus andtherefore lacked the sites for attachment of palmitate. The trun-cated PLC-b3 lacked the coiled-coil domain, a region of �300residues that is unique to the PLC-b family. The affinity of thetruncated PLC-b3 for binding to Gaq was found to be similar tothat of the full length enzyme. It was therefore concluded thatthe role of the coiled-coil domain was to mediate membraneassociation, that is, indirect regulation. This conclusion howeveris clouded by two inconvenient truths. First, dependence on thecoiled-coil domain is specific to Gaq. This domain is not requiredfor stimulation of lipase activity by Gbc subunits, Rac or Ca21.Second, isolated coiled-coil domain fragments exhibit GTPaseactivity for Gaq (9). Therefore, a second interaction site with Gaq

must exist within the coiled-coil domain.Indeed, the recent structure of full-length PLC-b3 in com-

plex with activated Gaq reinstated the central role of thecoiled-coil domain in regulation, independent of binding (18).The coiled-coil domain, specifically a hydrophobic patch of theDa5 helix, was shown to directly interact with the N-terminal

a-helical domain of Gaq during catalysis and was required forfull efficacy. Perturbation of the hydrophobic patch in Da5resulted in a marked reduction in Gaq-stimulated lipase activ-ity despite an intact membrane-binding surface on the coiled-coil domain. Gaq palmitoylation was found to be necessary forfull efficacy (18). The coiled-coil domain also interacted withthe core structure of PLC-b3.

Interaction between the coiled-coil domain and the N-terminal a-helical domain of Gaq strategically positions PLC-bGAP to mediate kinetic coupling. All Ga subunits share a simi-lar tertiary structure composed of two domains, a Ras-likeGTPase domain and a-helical domain (19). The Ras-likedomain is conserved among all members of the G proteinsuperfamily. This domain contains the site of GTP hydrolysisand binding sites for Gbc, receptor and effectors. TruncatedPLC-b3 interacted with this domain (17).

The Ga helical domain however is unique to the heterotri-meric Ga subunit and appears crucial for G protein/activatedreceptor interaction (19). The Ga helical domain covers theguanine nucleotide binding site of the Ras-like domain. Struc-tural data of Gs in complex with ligand-bound b2 adrenergicreceptor showed that the a-helical domain undergoes amarked displacement from the Ras-like domain exposing thenucleotide binding pocket (20,21). PLC-b1 GAP was shown toincrease the rate of dissociation of bound-GDP, the rate limit-ing step in G protein activation (8).

Context-Dependent Regulation ofPLC-b1 GAP FunctionWe demonstrated that the novel phospholipid mediator, phos-phatidic acid (PA) regulated Gq efficacy (22,23). PA couldincrease Gq efficacy by stimulating PLC-b1 GAP. Regulation byPA depended on signaling via the unique PLC-b1 PA bindingdomain that we localized to the Gaq binding region of the coiled-coil domain (24,25). Residues required for stimulation wereunique to PA and did not overlap with residues necessary forstimulation by Gaq or Gbc, or for membrane-association (22,26).The PLC-b1 PA binding region is unique to the PLC-b1 isoform.In short, PLC-b1 appears programmed for regulation by PA.

Building on all this knowledge, a new hypothesis is pro-posed that could unify the two conflicting paradigms for PLC-b1 GAP function, as it pertains to physiological signaling andnative levels of protein (Fig. 1). In the absence of ligand, PLC-b1 GAP activity could indeed function to deactivate Gq signal-ing. PLC-b1 GAP would determine the threshold for stimula-tion, that is, the ligand concentration at which a response isdetected. What would be the physiological advantage to thisfunction? PLC-b1 GAP would limit spurious or leaky Gq signal-ing to sharpen signaling kinetics. Many GPCRs have beenfound to exhibit basal or ligand independent activity thatallows them to activate G proteins (27).

At threshold, the ligand-mediated increase in GPCR GEFactivity increases the fraction of Gaq-GTP. PA would stimulate

Litosch 937

PLC-b1 GAP activity to increase signaling efficiency as depend-ent on the PLC-b1 PA binding domain. PA-stimulated PLC-b1

GAP would kinetically couple (coordinate) with GPCR GEF toincrease signal amplitude and signal strength, that is, Gq

efficiency.In vitro assays using purified proteins reconstituted at con-

trolled concentrations into phospholipid vesicles have shownthat PLC-b1 is a very good GAP (4,5). PLC-b1 GAP accelerateshydrolysis of Ga-GTP by about 1000-fold (5). Furthermore,kinetic coupling was observed in the absence of PA. HoweverPLC-b1 GAP activity in cells is not known and cannot beextrapolated from the in vitro data. Protein concentrations ofGPCR, Gq, and PLC-b1 at the membrane and regulated associa-tion of these proteins is not known (8,15).

Intriguingly, context-dependent regulation of PLC-b1 GAPfunction could contribute to Gq efficacy through Gbc (Fig. 2).Gbc signals independently of Ga but how Gbc achieves speci-ficity is unclear (28,29). It is thought that the Gbc releasedfrom activation of Gi/o is exclusively responsible for allresponse. However, Gq could regulate localized signalingthrough its pool of Gbc as dependent on PA-regulation andkinetic coupling.

The two major pathways that lead to an increase in levelsof PA are the mammalian phospholipase D (PLD1, PLD2) (30)and the large family of diacylglycerol kinases (DGK; (31)). Weidentified RhoA-regulated PLD1 and DGKf in the regulation ofGq efficacy in transfected COS-7 cells (23,32). DGKf phospho-

rylates PLC-generated DAG to PA and therefore could regulateresponse through a localized increase in PA levels or inhibitionof negative feedback regulation by protein kinase C.

A model that could unify two paradigms for regulation of Gq efficacy by PLC-b1 GAP. In the absence of ligand (constitutive

activity), PLC-b1 GAP accelerates Gaq GTPase activity to prevent leaky activation to set the threshold for stimulation to sharpen

signaling kinetics. At threshold, the increase in levels of PA is proposed to stimulate PLC-b1 GAP to kinetically couple with

GPCR GEF to increase Gq efficacy, as dependent on signaling via the unique PLC-b1 PA binding domain. The function of PLC-b1

GAP would therefore depend on PA, as determined during physiological signaling at native levels of protein. [Color figure can

be viewed in the online issue, which is available at wileyonlinelibrary.com.]

PLC-b1 GAP could regulate Gbc signaling as dependent

on PA signaling via the unique PLC-b1 PA binding domain.

Gbc subunits associated with the Gq heterotrimer could

coordinate with Gaq-GTP to orchestrate a complex

response. [Color figure can be viewed in the online issue,

which is available at wileyonlinelibrary.com.]

FIG 1

FIG 2

IUBMB LIFE

938 REGULATION OF PLC-b1 GAP FUNCTION by PA

PLC-b1 GAP and the Connection toCo-signaling with G12/13

GPCRs are classically depicted as monomers that signal witha single G protein subfamily. G protein subfamilies aregrouped by their Ga subunit based on sequence identity andeffectors (33). GPCRs, however, can co-signal with variousG proteins (34,35). Muscarinic receptors co-signal with Gq

and G12/13 (G12). The protease-activated receptor 1 (PAR1)co-signals with Gq, G12, Gs, and Gi. Many intriguing ques-tions surround the mechanistic basis for G protein co-signal-ing. Does a GPCR signal independently with different G pro-teins? Could co-signaling depend on GPCR heterodimers?Does each G protein regulate a unique aspect of theoverall response or do they coordinate to shape GPCRpharmacology?

A GPCR could co-signal by engaging different G proteinsthrough unique binding sites. Mutagenesis showed that PAR1uses different residues within the second intracellular loop tocouple to G proteins (36). Replacement of five amino acids

individually within the PAR1 i2 loop inhibited Gq but not G12 orGi signaling.

Co-signaling could depend on GPCR heterodimers. GPCRscan form transient heterodimers (37,38). Dimerization of therhodopsin class A GPCRs does not appear to be important forreceptor function (39). Heterodimerization however may shapereceptor pharmacology by altering ligand-binding properties,efficiency of G protein activation and selective engagement ofdownstream signaling pathways.

The herein proposed context-dependent regulation of PLC-b1 GAP function by PA suggests a mechanism for regulation ofGq efficacy by co-signaling as mediated by GPCR monomers ortransient heterodimers (Fig. 3). In both scenarios, PA is sup-plied from ligand-activated GPCRs, coupled to G12-RhoA-PLD1signaling, to stimulate PLC-b1 GAP to kinetically couple withGPCR GEF to increase Gq efficiency.

Lipidomics has revealed that cells generate many differentspecies of lipids (40). Regulation of PLC-b1 GAP function coulddepend on the species of PA and their spatiotemporal dynam-ics. We do not know whether PA could be a common

A model that could explain how context-dependent regulation of PLC-b1 GAP function by co-signaling would determine GPCR

pharmacology. In scheme (a) co-signaling is mediated by ligand-activated GPCR monomers that are coupled to either Gq or

G12. Kinetic coupling would depend on the spatial stability of the PA signal. In scheme (b) a GPCR heterodimer is shown as the

basic signaling unit. Relative to the monomer, kinetic coupling could be stabilized because of the localized PA signal. The

increase in levels of PA, from the activated GPCR coupled to RhoA-PLD1 signaling, would stimulate PLC-b1 GAP to kinetically

couple with GPCR GEF to increase Gq efficacy as dependent on the unique PLC-b1 PA binding domain. Co-signaling could

therefore increase Gq-efficiency relative to a GPCR that only signals with Gq. [Color figure can be viewed in the online issue,

which is available at wileyonlinelibrary.com.]

FIG 3

Litosch 939

mechanism to regulate PLC-b GAP activity and kinetic cou-pling. PA was shown to stimulate PLC-b3 lipase activity (25).

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940 REGULATION OF PLC-b1 GAP FUNCTION by PA